This invention is generally in the field of methods and compositions for the determination and quantitation of lipoproteins and apolipoproteins in human blood.
Human Plasma Lipoproteins and Apolipoproteins
Plasma lipoproteins are carriers of lipids from the sites of synthesis and absorption to the sites of storage and/or utilization. Lipoproteins are spherical particles with triglycerides and cholesterol esters in their core and a layer of phospholipids, nonesterified cholesterol and apolipoproteins on the surface. They are categorized into five major classes based on their hydrated density as very large, triglyceride-rich particles known as chylomicrons (less than 0.95 g/ml), very low density lipoproteins (VLDL, 0.95 to 1.006 g/ml), intermediate-density lipoproteins (IDL, 1.006 to 1.019 g/ml), low-density lipoproteins (LDL, 1.019 to 1.063 g/ml) and, high-density lipoproteins (HDL, 1.063 to 1.210 g/ml). Plasma lipoproteins can be also classified on the basis of their electrophoretic mobility. HDL comigrate with .alpha.-globulins, LDL with .beta.-globulins, VLDL between .alpha.- and .beta.-globulins with so called pre-.beta. globulins, whereas chylomicrons remain at the point of application. (Osborne, J. D. and Brewer, B. Jr. Adv. Prot. Chem. 31:253-337 (1977); Smith, L. C. et al. Ann. Rev. Biochem., 47:751-777 (1978)).
Apolipoproteins are protein components of lipoproteins with three major functions: (1) maintaining the stability of lipoprotein particles, (2) acting as cofactors for enzymes that act on lipoproteins, and (3) removing lipoproteins from circulation by receptor-mediated mechanisms. The four groups of apolipoproteins are apolipoproteins A (Apo A), B (Apo B), C (Apo C) and E (Apo E). Each of the three groups A, B and C consists of two or more distinct proteins. These are for Apo A: Apo A-I, Apo A-II, and Apo A-IV, for Apo B: Apo B-100 and Apo B-48; and for Apo C: Apo C-I, Apo C-II and Apo C-III. Apo E includes several isoforms.
Each class of lipoproteins includes a variety of apolipoproteins in differing proportions with the exception of LDL, which contains Apo B-100 as a sole apolipoprotein. Apo A-I and Apo A-II constitute approximately 90 percent of the protein moiety of HDL whereas Apo C and Apo E are present in various proportions in chylomicrons, VLDL, IDL and HDL. Apo B-100 is present in LDL, VLDL and IDL. Apo B-48 resides only in chylomicrons and so called chylomicron remnants (Kane, J. P., Method. Enzymol. 129:123-129 (1986)).
Lipoprotein metabolism is a very complex process involving exogenous and endogenous pathways as well as a reverse cholesterol transport. In the exogenous pathway, the triglycerides and cholesterol from an individual's diet are incorporated into chylomicrons which enter into the blood stream via intestinal lymph. Lipoprotein lipase hydrolyzes the triglyceride component of chylomicrons into free fatty acids which are taken up by muscle cells and/or adipocytes. As the triglyceride core of chylomicrons is depleted, chylomicron remnant particles are formed and removed from the circulation via chylomicron remnant receptor present on the surface of hepatic cells.
In the endogenous pathway, the liver synthesizes triglycerides and cholesterol. The endogenously made triglycerides and cholesterol are packed into triglyceride rich VLDL particles and secreted into the circulation. Once in the blood, most of the triglyceride content of VLDL particles is hydrolyzed by lipoprotein lipase, releasing free fatty acids to be used as a source of energy or for storage. As a result of this process, VLDL particles diminish in size and increase in density and are converted into VLDL remnants or IDL. Further processing includes additional lipolysis and exchange of lipids and apolipoproteins between IDL and HDL, leading to the formation of LDL which contain mostly cholesterol esters in the core and phospholipids and Apo B-100 on the surface. LDL particles are taken up by the hepatic and extrahepatic cells via specific LDL-receptor.
The reverse cholesterol transport pathway starts with the secretion of nascent HDL particles which are produced by the liver and intestine. These disk-like particles consist primarily of phospholipids surrounded by Apo A-I. They accept free cholesterol from peripheral tissues which is esterified and translocated into the core of HDL particles, which become spherical and ready to deliver their cholesterol content to hepatocytes. During the degradation of VLDL and LDL, HDL particles also accept free cholesterol and apolipoproteins from these lipoproteins.
Role of Lipoproteins in Atherosclerosis
Atherosclerosis is a chronic disease characterized by progressive deposition of cholesterol, fibrous elements and minerals in arterial walls. Atherosclerosis is the underlying pathophysiological process of coronary heart disease (CHD), one of the leading causes of death in Western World (Report of the Working Group on Atherosclerosis of the National Heart and Lung and Blood Institute, 2 (Washington, D.C.: Government Printing Office, 1981) DHEW Publication No. (NIH) 82-2035). Although development of CHD is a very complex process influenced by many contributing factors, subintimal cholesterol deposition in coronary arteries is one of the earliest and most important events during the course of disease. The major source of cholesterol found in arterial wall deposits is plasma lipoproteins. Because of their diverse metabolic roles and properties, lipoproteins associate differently with the risk of developing CHD.
LDL particles constitute approximately two-thirds of total cholesterol (TC) and form the primary atherogenic fraction of the serum cholesterol. Many epidemiological and clinical studies have shown that increased LDL levels in the blood are associated with an increased risk of CHD. For example, the results of the Lipid Research Clinics trial have shown that reduction of LDL-cholesterol (LDL-C) is associated with a significant decrease in CHD incidence (The Lipid Research Clinics Coronary Primary Prevention Trial results:II. JAMA 251:365-374 (1984)).
The evidence relating CHD and triglyceride-rich lipoproteins such as VLDL is not as strong as for the LDL. Many studies have shown a positive correlation between elevated serum triglyceride levels and increased risk of CHD. However, the independent link between elevated serum triglyceride (TG) and CHD breaks down when multivariate analyses are used to control statistically for the effects of total cholesterol (TC) and HDL-cholesterol (HDL-C). These observations suggest that increased CHD risk noted in patients with hypertriglyceridemia could be due to either the accumulation of triglyceride-rich particles that are uniquely atherogenic in some people or to the association with reduced HDL-C (Assmann, G. et al., Am. J. Cardiol., 68:1-3 (1991)). Remnants of triglyceride-rich particles, (for example, chylomicron and VLDL remnants) which are found in IDL are also atherogenic (Krauss, R. M., Am. Heart J., 112:578-582 (1987)).
In contrast to the atherogenic potential of LDL, VLDL and VLDL remnants, HDL are inversely correlated with CHD, so that individuals with low concentrations of HDL-C have an increased incidence of CHD (Gordon, T. et al., Am. J. Med., 62:707-714 (1977); Miller, N. E. et al., Lancet, 1:965-968 (1977); Miller, G. J. and Miller, N. E., Lancet, 1:16-19 (1975)). At the other extreme, individuals with high concentrations of HDL, such as found in familial hyperalphalipoproteinemia, seldom express symptoms of CHD. The fact that pre-menopausal females have higher HDL concentrations and less CHD compared to males, also supports the anti-atherogenic role of HLD. Furthermore, postmenopausal women have a significant increase in CHD risk while their HDL concentrations decrease.
Measurement of LDL
LDL consists of a hydrophobic lipid core composed of cholesterol esters and triglycerides. The lipid core of the LDL particle is surrounded by an amphipathic coat composed of phospholipids, unesterified cholesterol and Apo B. Each LDL particle contains one molecule of Apo B-100. On a weight basis, LDL is composed of 38 percent cholesterol ester, 22 percent phospholipid, 21 percent protein, 11 percent triglyceride and 8 percent unesterified cholesterol.
Accurate measurements of LDL using presently available technology depends on separation of LDL particles from other lipoproteins. Once the LDL particles are separated, their concentration can be quantified by determination of their cholesterol (LDL-C) or Apo B (LDL-B) content. LDL-C is the most commonly used parameter.
Several ultracentrifugation methods have been developed over the years to separate serum lipoproteins. Analytical ultracentrifugation was developed in the 1950s and continues to be used today in some research laboratories. In this technique, lipoproteins are separated by analytical ultracentrifugation and quantitated by optical refraction. This method of quantitation measures lipoprotein mass, but does not give any information about lipid or protein composition. Sequential ultracentrifugation was developed in 1955 to overcome some of the limitations of analytical ultracentrifugation. In this technique, lipoproteins are separated by repeated ultracentrifugations after progressively increasing the sample density. Lipoproteins can be isolated within any desired density interval and in sufficient quantities to allow for multiple chemical analyses. Sequential ultracentrifugation continues to be used today for preparative isolation of lipoproteins. However, the ultracentrifugation methods are too expensive and time consuming for the purpose of measuring LDL-C levels to assess lipoprotein abnormalities and CHD risk in routine clinical application. Other methods for separating LDL include size-exclusion and other types of chromatography, electrophoresis, and precipitation. The size-exclusion chromatography methods include agarose column chromatography and high-performance gel filtration column chromatography. The time required for analysis, typically 24 hours, is the major difficulty with agarose column chromatography. The development of high-performance liquid chromatography (HPLC) methods has reduced the analysis time, but has increased the cost and complexity of the procedure. Affinity chromatography using anti-LDL antibodies, heparin, or dextran sulfate linked to SEPHAROSE.TM. (Pharmacia LKB, Piscataway, N.J.) gels has also been used to isolate LDL.
Electrophoresis methods, which separate lipoproteins according to their charge in addition to size, have been used in many clinical laboratories. This technique is helpful in qualitative assessment of various types of hyperlipoproteinemias. Agarose gel electrophoresis at pH 8.6, followed by visualization using lipophilic stains such as Oil Red O, Sudan Black B or Sudan Red 7B, have been commonly used with commercial reagents packaged as kits, for example, as sold by Ciba Corning (Medfield, Mass.) Lipoprotein concentrations are then estimated by densitometry based on the color intensity of the separated bands.
Several methods for selective chemical precipitation of LDL have been described and commercialized (Mulder, K. et al., Clin. Chim. Acta 143:29-35 (1984)). The precipitation methods, which quantitate LDL-C as the difference between the total cholesterol and the sum of VLDL- and HDL-cholesterol (Friedewald, W. T. et al., Clin. Chem. 18:499-502 (1972)), are precise and produce reasonably accurate results relative to ultracentrifugation methods when serum TG values are low. However, most investigators have found that the precipitation methods are plagued with systematic errors when samples with high TG levels are analyzed.
Most recently, a method was developed which uses latex beads coated with affinity-purified polyclonal goat antisera directed against apolipoproteins in HDL and VLDL (Sigma, St. Louis, Mo.). In this method, a plasma or serum sample is incubated with the beads for 5 to 10 minutes at room temperature and then centrifuged for 5 minutes to remove the HDL and VLDL bound to the beads. The remainder of the sample is then assayed for cholesterol using a standard enzymatic cholesterol assay (Sigma St. Louis, Mo.) to obtain a value for the LDL-C, the presumed remaining source of cholesterol in the sample.
Techniques used for measurement of LDL by its Apo B content include radioimmunoassay; enzyme immunoassay (ELISA competitive or capture systems), fluorescence immunoassay, radial immunodiffusion, nephelometry, turbidimetry and electroimmunoassay.
The National Cholesterol Education Program (NCEP) recommended the determination of LDL-C concentration in diagnosis and treatment of hypercholesterolemia. According to NCEP, concentrations lower than 130 mg/dl in adults are considered desirable, concentrations between 130 and 150 mg/dl are borderline high, and concentrations above 160 md/dl are high (see Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults, Arch. Intern. Med. 148:36-69 (1988)). The NCEP also recommended determining LDL-cholesterol concentrations for children and adolescents since the high LDL correlates with the extent of coronary and aortic atherosclerosis in this age group as well as development of CHD later in life. Cholesterol values of 110 mg/dl are desirable, values between 110 and 129 mg/dl are borderline high, and values above 130 mg/dl are considered high in children and adolescents (see Report of the Expert Panel on Blood Cholesterol Levels in Children and Adolescents, Pediatrics 89:525-584 (1992)).
Measurement of HDL
HDL, the smallest in size of the lipoproteins, includes a family of lipoprotein particles that exist in a constant state of dynamic flux as they interact with LDL, IDL and VLDL. HDL have the highest proportion of protein (50 percent) relative to lipid compared to other lipoproteins. The major HDL proteins are Apo A-I and Apo A-II, with lower concentrations of Apo C(I,II & III), E, and A-IV. Phospholipids are the principal lipid component of HDL, with cholesterol esters, unesterified cholesterol, and TG present in lower concentrations.
As in the case of LDL, HDL is typically measured after its separation from other lipoproteins and quantification of cholesterol in the HDL (HDL-C). As described above, the separation of HDL can be accomplished by ultracentrifugation, chromatographic procedures, electrophoresis and precipitation. The reliability of lipoprotein quantitations following separation by ultracentrifugation techniques depends upon both the performance of the analytical quantitation method, such as cholesterol analysis, and the skills of the technologist in performing accurate recovery and transfer of the lipoprotein fractions from the ultracentrifuge tube. HDL-C is more easily quantitated by selective precipitation techniques compared to either ultracentrifugation or electrophoretic methods. Currently, the majority of clinical laboratories use either dextran sulfate or sodium phosphotungstate procedures for HDL-C analysis (Warnick, G. R. et al., Clin. Chem. 28:1379-1388 (1982); Lopes-Virella, M. F. et al., Clin. Chem. 23:882-884 (1977)). According to NECP guidelines, patients with HDL-C levels below 35 mg/dl are considered to be at risk for CHD.
LDL/HDL Ratio
Some studies have demonstrated that the ratio between LDL-C and HDL-C represents a better predictor of CHD than either of these two parameters alone (Arntzenius, A. C., Acta. Cardiol., 46:431-438 (1991); Barth J. D. and Arntzenius, A. C., Eur. Heart J., 12:952-957 (1991); Ortola, J. et al., Clin. Chem., 38:56-59 (1992); Gohlke H., Wien Klin. Wochenschr., 104:309-313 (1992)).
LPA-I and LPA-I:A-II Lipoprotein Particles There are two subpopulations of HDL lipoprotein particles known as LPA-I and LPA-I:A-II (Koren, E. et al. Clin. Chem., 33:38-43 (1987)). LPA-I particles contain Apo A-I but no Apo A-II while LPA-I:A-II particles contain both apolipoproteins. These HDL subpopulations can be measured by enzyme immunoassay (Koren, E. et al. Clin. Chem., 33:38-43 (1987)) or electroimmunoassay (Atmeh, R. F. et al., Biochim. Biophys. Acta, 751:175-188 (1983)). Their importance has been emphasized by several studies which demonstrated that LPA-I is a more active component in reverse cholesterol transport and, therefore, more anti-atherogenic than other lipoproteins (Puchois, P. et al., Atherosclerosis, 68:35-40 (1987); Fruchart, J. C. and Ailhaud, G., Clin. Chem., 38:793-797 (1992)).
Measurements of VLDL. IDL, C-III and E Ratios
Triglyceride-rich VLDL as well as their remnants (IDL) can be separated by the above ultracentrifugational, chromatographic and electrophoretic methods and quantified by determination of their cholesterol content. Although atherogenic, these lipoprotein particles are not commonly measured in routine clinical laboratories. Instead, serum triglyceride concentration in the fasting state is considered representative of the VLDL content and is used traditionally in the assessment of the VLDL-related CHD risk. More recently, measurements of the so-called C-III and E ratios have been proposed as reliable predictors of the VLDL-related CHD risk. The principle of these measurements is to precipitate all Apo B-100-containing particles (VLDL, IDL and LDL) with heparin which leaves HDL in the heparin supernate. This separation is followed by an immunochemical determination of Apo C-III or Apo E in the heparin precipitate and heparin supernate and calculation of the corresponding ratios by dividing C-III or E concentration in heparin supernate with their respective concentrations in heparin precipitate (Alaupovic, P., Can. J. Biochem., 59:565-579 (1981)). Most of the Apo C-III and Apo E in the heparin precipitate is associated with Apo B in VLDL and VLDL remnant (IDL) particles. The C-III and E in the heparin supernate is associated with Apo A-I in HDL particles. Apo C-III and/or Apo E in the heparin precipitate reflects the concentration of VLDL and VLDL-remnant particles both of which are atherogenic. The Apo C-III and Apo E in the heparin supernate represents HDL particles which are anti-atherogenic. Therefore, a low C-III and E ratio is associated with increased risk of CHD because it reflects either high VLDL and IDL and normal HDL or, more frequently, high VLDL and IDL combined with low HDL. In fact, the predictive power of C-III ratio has surpassed that of triglycerides in several clinical studies (Alaupovic, P. and Blankenhorn, D. H., Klin. Wochenschr., 60:38-40 (1990); Blankenhorn, D. H. et al., Circulation 81:470-478 (1990)).
Measurements of Apo A-I and B
Apo B-100 is an integral component of the four major atherogenic lipoproteins: VLDL, IDL, LDL and Lp(a). Apo B-100 is distinguished from Apo B-48, which is found only in lipoproteins of intestinal origin, such as chylomicrons and chylomicron remnants. Apo B-48 is usually undetectable in the systemic circulation, except in rare subjects with Type I, III, or V hyperlipidemia. Apo B's initial function in VLDL and IDL appears to be structural; however, with exposure of binding domains on LDL, it becomes responsible for interaction with high-affinity LDL receptors on cell surfaces, which results in uptake and removal of LDL from the circulation. Several studies have shown that an increased Apo B level in blood is a reliable marker for coronary atherosclerosis (Sniderman, A. et al., Proc. Natl. Acad. Sci. USA, 77:604-608 (1980); Kwiterovich, P. O. et al., Am. J. Cardiol., 71:631-639 (1993); McGill et al. Coron. Artery Dis., 4:261-270 (1993); Tornvall, P. et al., Circulation, 88:2180-2189 (1993)).
Apo A-I is the major protein constituent of lipoproteins in the high density range. Apo A-I may also be the ligand that binds to a proposed hepatic receptor for HDL removal. A number of studies support the clinical sensitivity and specificity of Apo A-I as a negative risk factor for atherosclerosis (Avogaro, P. et al., Lancet, 1:901-903 (1979); Maciejko, J. J. et al., N. Enql. J. Med., 309:385-389 (1983)). Some investigators have also described Apo A-I/Apo B ratio as a useful index of atherosclerotic risk (Kwiterovich, P. O. et al., Am. J. Cardiol., 69:1015-1021 (1992); Kuyl, J. M. and Mendelsohn, D., Clin. Biochem., 25:313-316 (1992)).
Techniques used for both Apo A-I and B are confined to immunological procedures using antibodies directed against Apo A-I or B and include radioimmunoassay (RIA), enzyme immunoassay (ELISA), competitive or capture systems, fluorescence immunoassay, radial immunodiffusion, nephelometry, turbidimetry and electroimmunoassay.
To summarize, there are several lipoprotein related parameters that are currently used as predictors of CHD. Some of them represent atherogenic lipoproteins (total cholesterol, triglycerides, LDL, IDL, VLDL, Lp(a) and Apo B and are positively associated with CHD whereas the others are antiatherogenic factors, HDL, Apo A-I and LPA-I which are inversely related to the disease. The ratios of some of these parameters, such as LDL/HDL, Apo A-I/Apo B, C-III and E ratio, appear to be even more sensitive predictors of CHD because each of them reflects both anti-atherogenic and atherogenic factors in a single parameter.
All of the methods currently used to determine lipoprotein related risk factors require a laboratory with the necessary equipment and trained personnel to carry out each of the technical steps, to perform the necessary calculations and to interpret the results. The only exception is a new total cholesterol measurement device (AccuMeter Cholesterol Self-Test) developed by ChemTrack (Sunnyvale, Calif.) and designed for home use. However, a total cholesterol level is a less sensitive predictor compared to the levels of specific lipoproteins, apolipoproteins or ratios thereof.
It is therefore an object of the present invention to provide methods and means to rapidly and reliably determine levels of specific lipoproteins, apolipoproteins or the ratios thereof in whole blood, serum or plasma without the necessity of laboratory equipment or technically trained personnel.
It is another object of the present invention to provide antibodies immunoreactive with specific epitopes on lipoproteins, such as those on LDL, VLDL and HDL, that enable rapid and reliable determinations of levels of lipoproteins and/or apolipoproteins in whole blood, serum or plasma.